Targeting apoptosis by the remodelling of calcium-transporting proteins in cancerogenesis

Authors

  • Charlotte Dubois,

    1. Inserm, U-1003, Equipe labellisée par la Ligue Nationale contre le cancer. Laboratory of Excellence, Ion Channels Science and Therapeutics, Université des Sciences et Technologies de Lille (USTL), Villeneuve d'Ascq, France
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    • These authors contributed equally to this work
  • Fabien Vanden Abeele,

    1. Inserm, U-1003, Equipe labellisée par la Ligue Nationale contre le cancer. Laboratory of Excellence, Ion Channels Science and Therapeutics, Université des Sciences et Technologies de Lille (USTL), Villeneuve d'Ascq, France
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    • These authors contributed equally to this work
  • Natacha Prevarskaya

    Corresponding author
    1. Inserm, U-1003, Equipe labellisée par la Ligue Nationale contre le cancer. Laboratory of Excellence, Ion Channels Science and Therapeutics, Université des Sciences et Technologies de Lille (USTL), Villeneuve d'Ascq, France
    • Correspondence

      N. Prevarskaya, Inserm, U-1003, Equipe labellisée par la Ligue Nationale contre le cancer, Villeneuve d'Ascq, France

      E-mail: natacha.prevarskaya@univ-lille1.fr

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Abstract

Calcium is a universal messenger regulating many physiological functions, including the ability of the cell to undergo orderly self-destruction upon completion of its function, called apoptosis. In physiopathological conditions such as cancer, apoptotic processes become deregulated, leading to apoptosis-resistant phenotypes. Recently, perturbations of cellular calcium homeostasis have been described in apoptosis-resistant cell phenotypes. Thereby, new molecular actors have been identified, offering more accurate research possibilities in the field of apoptosis resistance and providing the bases for more rational approaches to cancer treatments. In this review, we focus on the calcium-transporting protein-dependent pathways involved in apoptosis, which are deregulated by cancer. We present the calcium-transporting proteins involved in the deregulation of apoptosis, and those chemotherapies that target actors in calcium-induced apoptosis.

Abbreviations
[Ca2+]ER

endoplasmic reticulum calcium concentration

[Ca2+]i

intracellular calcium concentration

4-AP

4-aminopyridine

ER

endoplasmic reticulum

IP3R

inositol 1,4,5-trisphosphate receptor

PMCA

plasma membrane Ca2+-ATPase

ROS

reactive oxygen species

SERCA

sarcoplasmic/endoplasmic reticulum Ca2+-ATPase

SOCE

store-operated channel entry

TRP

transient receptor potential

TRPP2

transient receptor potential polycystic 2

Introduction

Cancer is caused by defects in the mechanisms underlying cell proliferation and cell death. The development of tumours results from excessive cell proliferation combined with inhibition of cell apoptosis, which eventually leads to imbalances in tissue homeostasis and uncontrolled proliferation [1]. Proliferation and apoptosis involve different pathways and molecular actors, proliferation relying on cyclin-dependent protein kinases – regulators of the cell division cycle [2] – and apoptosis primarily depending on caspases – cysteine proteases executing a cell death programme [3]. Nevertheless, calcium ions are central to both phenomena, serving as major agents of calcium signalling, ultimately determining the cell's fate. From these observations, it is clear that changes in the free intracellular calcium concentration ([Ca2+]i) alone are insufficient for governing such diverse processes deciding cell fate. Therefore, the amplitude, spatial localization and temporal characteristics of calcium signals are of major importance in determining death, survival, and proliferation [4, 5]. The formation of local signalling complexes owing to restricted calcium diffusion [6] and the involvement of molecular actors (i.e calcium channels, associated proteins, ion exchangers, etc.) may well allow even greater specialization of the cellular responses controlled by calcium. Well-documented reviews describing the development of tumours and other calcium-dependent pathways, such as migration [7, 8], differentiation [9, 10], and proliferation [11, 12], have been published by specialists. In-depth analysis of the complex apoptotic machinery in physiological conditions has already been presented in a number of recent specialized reviews [13-17]; therefore, we restrict ourselves to outlining those actors involved in the failure of calcium-dependent apoptosis that are observed in various cancer models.

Apoptosis is an orderly physiological process that allows the elimination of cells that have already completed, or for any reason are incapable of performing, their physiological function, and are therefore no longer necessary. It is important for normal embryonic development and for the maintenance of tissue homeostasis in the adult organism. Imbalances in apoptosis can cause diseases such as cancer. The calcium dependence of apoptosis has been well defined and comprehensively described in numerous reviews [13, 18, 19]. Calcium apoptotic signalling involves two critical factors – sustained elevation of [Ca2+]i, and a prolonged decrease in the calcium concentration in the endoplasmic reticulum ([Ca2+]ER) with variable contributions of these factors, depending on cell type. Thus, to effectively evade apoptosis, cancer cells must employ mechanisms that do two things. First, they must substantially reduce or even prevent calcium influx by downregulating the expression of calcium-permeable channels or the signalling pathways that lead to their activation. Second, they must enable them to adapt to the chronic underfilling of the endoplasmic reticulum (ER) calcium store [18, 20]. Reduced calcium influx in cancer cells prevents calcium overload in response to proapoptotic stimuli, thereby impairing the effectiveness of mitochondrial and cytoplasmic apoptotic pathways, whereas adaptation to the reduced ER calcium content diminishes activation of ER stress-dependent responses.

In this review, we discuss the remodelling of calcium signalling actors involved in the defect of apoptosis observed during the progression of various cancers, and the potential therapies based on calcium pathways that are either used in the clinic or are currently under development.

Calcium-transporting protein-induced apoptosis is deregulated during cancer

The entry to the cell

It is generally accepted that intracellular calcium signals destined to support life processes such as proliferation, differentiation, migration and secretion have a dynamic nature and are structured in space and time, often taking the form of calcium oscillations, waves, sparks, spikes, flickers, etc. [21, 22]. Calcium is toxic to the cell, and for this reason the basal intracellular concentration of calcium is usually maintained at a very low level of ~ 100 nm, as compared with an extracellular concentration of 1.2 mm. To achieve this, cells need different effectors, such as pumps, ion channels or exchangers, to maintain the gradient [23].

Such complex patterns are generated by coordinated interactions between calcium entry, calcium mobilization, calcium extrusion and calcium uptake mechanisms, under the control of extracellular agonists and intracellular receptor-coupled second messenger systems. On the other hand, cell death is usually associated with a global, sustained calcium increase resulting from excessive calcium entry and mobilization, commonly accompanied by a prolonged decrease in the ER calcium content [1, 18]. Because cancer is associated with the altered endogenous expression of calcium-handling proteins and/or calcium-regulated effectors, this results in the disruption of normal calcium signalling and the deregulation of both life and death-related processes, ultimately leading to the development of certain malignant phenotypes.

Plasma membrane Ca2+-ATPases (PMCAs)

PMCAs actively extrude calcium from the cell, and are essential components for maintaining intracellular calcium homeostasis. There are four PMCA isoforms (PMCA1–4), and alternative splicing of the PMCA genes creates over 30 distinct isoforms, which are expressed differentially in various cell types in normal and diseased states [24]. Different studies have shown that they are affected in various cancer models. Changes in PMCA expression have been reported in several forms of cancer [25, 26]. In breast cancer cell lines, there is modest upregulation of PMCA1 mRNA as compared with cell lines derived from noncancerous tissue, and there is pronounced upregulation of PMCA2 mRNA in some breast cancer cell lines, such as ZR-75-1 and T47D cells [26, 27]. Breast cancer cell lines also tend to have lower levels of PMCA4 mRNA [28]. A recent study demonstrated that PMCA1 knockdown augmented necrosis mediated by the calcium ionophore ionomycin, whereas apoptosis mediated by the inhibition of Bcl-2 (which belongs to the antiapoptotic protein family activated by the intrinsic pathway) was enhanced by PMCA4 silencing. PMCA4 silencing was also associated with an inhibition of nuclear factor-κB nuclear translocation, and a nuclear factor-κB inhibitor phenocopied the effects of PMCA4 silencing in promoting cell death induced by the inhibition of Bcl-2 [29]. Reduced expression of another PMCA isoform, PMCA2, augments ionomycin-mediated cell death in SH-SY5Y neuroblastoma cells [30], whereas its overexpression in T47D breast cancer cells confers resistance to ionomycin-mediated cell death through the attenuation of [Ca2+]i responses [27]. The potential significance of this survival advantage is reflected in the poorer prognosis of breast cancer patients with elevated PMCA2 expression [27]. Baggot et al. have identified an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells that confers cell resistance to apoptosis [31]. Differentiation of HT-29 colon cancer cells is associated with upregulation of PMCA4, consistent with augmented calcium extrusion [32], whereas, in human colon cancer samples in the early stage of progression, with cells becoming less differentiated, PMCA4 transcription decreases significantly. It was concluded that variations in PMCA4 expression and the associated remodelling of calcium efflux in colon cancer cells provide a growth advantage, while avoiding apoptosis and conferring insensitivity to apoptotic stimuli [32].

Transient receptor potential (TRP) channels

Members of the mammalian TRP family of ion channels are ubiquitously distributed, and have an extraordinary diversity of gating mechanisms [33]. These enable them to mediate calcium entry in response to a variety of stimuli, including second messengers generated in response to surface receptor stimulation, mechanical stimuli coming from plasma membrane stretch, and the physical and chemical characteristics of the microenvironment.

TRPV6 is a highly calcium-selective channel protein expressed at the plasma membrane. Studies have shown that this channel is strongly expressed in advanced prostate cancer and significantly correlates with the Gleason > 7 grading, making it a strong marker of tumour progression and subsequent invasion of healthy tissues. Previous studies have shown that TRPV6 is involved in highly calcium-selective currents in prostate cells, and that it is tightly regulated by intracellular calcium concentrations [34-36]. During the progression of cancer, the expression of TRPV6 is increased. This upregulation of TRPV6 has been demonstrated by Lehen'kyi et al. to be involved in the resistance to apoptosis observed in hormone-sensitive prostate cancer cells [37].

The ORAI family

This family exists in three isoforms, Orai1, Orai2, and Orai3, which have been shown to be involved in cancerogenesis. In physiological conditions, Orai1 proteins are associated as homotetramers, and form a calcium channel activated by store depletion of the ER. They are activated in response to the surface receptor-stimulated mobilization of calcium from the ER stores, and thereby provide calcium for refilling of the ER store, as well as for signalling purposes. STIM1 is a single transmembrane domain protein that is mostly localized in the ER membrane, serving as a [Ca2+]ER sensor through its luminal EF-hand calcium-binding domain. Following a decrease in [Ca2+]ER, STIM1 redistributes into punctae close to the plasma membrane, where it can interact with the Orai1 plasma membrane calcium-permeable channel, thereby triggering its activation. In prostate cancer cells, previous studies have demonstrated the involvement of store-operated channel entry (SOCE) in apoptosis induction [38]. During cancerogenesis in prostate cancer cells, Flourakis et al. demonstrated that expression of the calcium-permeable Orai1 channel, expressed at the plasma membrane, is decreased. This modification of expression leads to the downregulation of SOCE (whose principal constituent is Orai1), which is responsible for the resistance to apoptosis [39].

In breast cancer cells, Orai3 can mediate calcium entry, contributing to [Ca2+]i. Faouzi et al. suggested that Orai3 participates in apoptosis resistance [40]. These studies suggested that the expression level and type of association of Orai protein could be important factors in the development of an apoptosis-resistant phenotype in cancerous cell models. A nonclassical event involving Orai1 has also been described. Khadra et al. reported that clustering of Orai protein could also lead to apoptosis resistance. It has been shown that CD95, the adaptor protein Fas-associated death domain protein, colocalizes with Orai1 and triggers Orai1-mediated localized calcium entry, thereby preventing the activation of death signalling pathways [41].

In the cell

After crossing the plasma membrane, calcium is free in the cytoplasm. Different possibilities exist: calcium could either be taken up again by the ER or by the mitochondria, or could activate calcium-dependent protein present in the cytoplasm.

Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)

The SERCA pump is the only calcium uptake mechanism of the ER, which is why its function is key to maintaining the ER luminal calcium content at an optimal level for both protein processing and calcium signalling purposes. In humans, three genes (ATP2A1, ATP2A2, and ATP2A3) generate multiple isoforms, owing to developmental or tissue-specific alternative splicing [42]. The most ubiquitous SERCA isoform is the ATP2A2-encoded SERCA2, which is represented by three splice variants, SERCA2a–c. Whereas SERCA2a and SERCA2c are primarily expressed in the heart, SERCA2b is present in all tissues, and is thought to be the major isoform of the ER calcium pump. SERCA3, which has six splice isoforms, is often found to be coexpressed with SERCA2b, but generally has a more limited tissue distribution.

Downregulation of SERCA2b expression has also been shown to accompany the transition of prostate cancer to the aggressive androgen-independent phenotype. This has been explained as one of the adaptive responses of androgen-independent prostate cancer cells against ER stress-induced apoptosis via the maintenance of lowered ER calcium filling [18, 20]. Thus, it seems that reduced SERCA pump expression, irrespective of the mechanisms involved (epigenetic influences, mutations, or altered activity of the transcription factors), is the common characteristic feature of all malignancies whereby cancer cells resist apoptosis [43].

In small cell lung cancer (H1339) and adenocarcinoma lung cancer (HCC) cell lines, although not in squamous cell lung cancer (EPLC) and large cell lung cancer (LCLC) cell lines, the ER calcium content is lower than in normal human bronchial epithelium.

Bergner et al. have shown that a reduced calcium content is correlated with reduced SERCA pump expression and a reduction in the expression of calreticulin, which buffers calcium within the ER. Both reductions participate in increased apoptosis resistance [44]. In addition to these factors, authors have highlighted, in this lung cancer model, the involvement of another molecular actor expressed on the ER, namely the inositol 1,4,5-trisphosphate receptor (IP3R).

IP3R

The primary calcium release channel on ER membranes is IP3R. Deletion of the gene encoding IP3R results in apoptosis defects in response to multiple stimuli. Bergner et al. have demonstrated that IPR3 expression increases and participates in the fall in [Ca2+]ER, which is responsible for the apoptosis defect [44]. Conversely, augmented IP3R levels are associated with increased cell death [45]. A mechanistic basis for altered IP3R function during apoptosis was revealed with the discovery that cytochrome c binds to IP3R early in the apoptotic process. This interaction blocks the calcium-dependent inhibition of IP3R function, resulting in increased calcium release from internal stores. An increase in calcium concentration in the cytoplasm and mitochondrial calcium overload lead to the activation of apoptosis.

TRP potential polycystic 2 (TRPP2)

The ion channel is mutated in autosomal dominant polycystic kidney disease, and protects cells from apoptosis by lowering [Ca2+]ER. ER-resident TRPP2 counteracts the activity of the sarcoendoplasmic Ca2+-ATPase by increasing ER calcium permeability. This results in diminished cytosolic and mitochondrial calcium signals upon stimulation of IP3Rs, as well as a reduction in calcium release from the ER in response to apoptotic stimuli. At present, there is still a lack of understanding of the role of TRPP2 in the regulation of ER homeostasis and its involvement in the induction of apoptosis or in the acquisition of a resistant apoptosis phenotype in cancer cells [46].

These actors are involved in the phenotype remodelling of cancer cells, and are responsible for the acquisition of an apoptotis-resistant phenotype. On the basis of this evidence, modifications of expression, activity or localization could be used as a way to restore apoptosis. However, these are not the only routes. Indeed, some molecular actors that are not deregulated during cancer progression should be examined for this purpose, because of their specific activity in calcium-dependent apoptotic pathways.

A well-illustrated example is the plasmalemmal Na+/Ca2+-exchanger, which normally extrudes calcium from the cell (forward mode), but is also able to bring calcium into the cell (reverse mode) under special conditions, such as intracellular sodium accumulation or membrane depolarization. It has been shown that the reverse exchanger mode can be responsible for the increases in [Ca2+]i in several cancer cell types in response to the inhibitors of the Na+/K+-ATPase pump, cardiac glycosides [47, 48], and in HCT116 human colon cancer cells in response to the inhibitor of protein kinase C and sphingosine kinase, N,N-dimethyl-d-erythro-sphingosine [49].

Calcium-transporting molecules as potential targets of chemotherapy

Despite compelling evidence concerning the disruption of calcium homeostasis in cancer cells leading to the promotion of some malignant phenotypes, as well as the identification of key calcium-transporting molecules with altered expression and/or function, the number of therapeutic approaches using calcium-transporting proteins as potential targets for chemotherapy is still limited, although some are promising.

Calcium channels and exchangers expressed on the plasma membrane as potential targets of chemotherapy

The entry of calcium into the cell is an important component of apoptosis induction. Targeting channels or exchangers involved in this calcium entry is a way to potentiate apoptosis in the cell (Fig. 1).

Figure 1.

Calcium channels, pumps and exchangers as potential targets of chemotherapy: how to avoid extrusion from the cell by PMCA and increase calcium entry by molecular actors involved in apoptosis resistance. To induce calcium-mediated apoptosis, multiple possibilities exist. One of them consists of increasing [Ca2+]i by actions on PMCAs. PMCAs actively extrude calcium from the cell, and are essential components for maintaining intracellular calcium homeostasis; their inhibition by chemical agents such as caloxins and platinum II complex can be used to inhibit extrusion from the cell. The second possibility is to potentiate the reuptake of calcium by the mitochondria. Mitochondria are important actors in the induction of apoptosis, and it is well established that their overload leads to the release of effector apoptosis actors. The use of nonsteroidal anti-inflammatory drugs (NSAIDs) or the inhibition of P2XR by 4-AP can induce an increase in the concentration of calcium in the cytoplasm. This calcium will be taken up again by mitochondria, or will directly activate caspases by the activation of calpain 9. The inhibition of the sodium/potassium pumps by cardiac glucosides induces the same response as described for P2XR. When oubain is used to inhibit sodium/potassium pumps, release of ROS is observed. Another possibility is to increase the calcium concentration in the mitochondria by modifying their permeability. TRPV6 can activate entry of calcium from outside the cell, inducing relocalization of BAX to the outer membranes of mitochondria, the consequence of which is an increase in mitochondrial membrane permeability. All of these strategies involve extracellular calcium to overload mitochondria, but it is possible to mobilize intracellular calcium. The ER is the main stock of calcium in the cell. Thapsigargin induces the depletion of the ER calcium store; this calcium can be taken up again by mitochondria, thereby leading to the activation of actors involved in apoptosis. In some CD95 cancerous models, the adaptor protein Fas-associated death domain protein (FADD) colocalizes with Orai1, and triggers Orai1-mediated localized calcium entry, thereby preventing the activation of death signalling pathways. The use of inhibitors of such interactions by monoclonal antibody (mAbs) or synthetic ligands (ADAM10 and CD95L) is necessary to induce apoptosis.

Calcium channels

Various calcium channels (i.e. TRP channels and store operated channels) are expressed on the plasma membrane, and allow the influx of calcium into the cell. SOCE is the main mechanism of calcium entry into in cells. Recent studies have shown that, in breast cancer cells, the interaction between CD95 (FasL) and Orai1 leads to resistance to apoptosis [41]. To overcome this deregulation, some teams have suggested the development of recombinant CD95L molecules or agonistic monoclonal antibody (mAbs) against TRAIL receptor for therapeutic exploitation, and some compounds are being tested in clinical trials in order to avoid such interactions as that between CD95 and Orai1 [50, 51].

Nonetheless, CD95L is tightly regulated, and future therapeutic approaches may stem from studies addressing its post-translational modifications, such as processing by ADAM10 and SPPL2a (proteases that generate soluble FasL fragments) [52, 53], in order to prevent the resistance to apoptosis induced by Orai1 calcium entry.

TRPV6 is a member of the TRP channel family, and is necessary to maintain calcium homeostasis in the cell. In gastric cancer cells, it has been demonstrated that TRPV6 can be activated by capsaicin (an organic compound extracted from chili peppers), and induces more apoptosis than in normal gastric cells, by increasing mitochondrial permeability through the activation of Bax and p53 in a c-Jun N-terminal kinase-dependent manner [54].

Plasma membrane exchangers

Recently, studies have shown the interesting clinical use in cancer therapy of increasing contractile force in patients with cardiac disorders by the use of cardiac glycosides such as oleandrin, ouabain, and digoxin. Cardiac glycosides are involved in the inhibition of the plasma membrane Na+/K+-ATPase, leading to alterations in intracellular potassium and calcium levels. These glycosides are also able to stimulate and sustain calcium increases and apoptosis in androgen-independent metastatic human prostate adenocarcinoma cells. Cell death is associated with the early release of cytochrome c from mitochondria, which is followed by proteolytic processing of caspases 8 and 3 [47].

Xu et al. showed that ouabain (inhibitor of Na+/K+-ATPase α1), in a hepatocellular carcinoma model, could induce apoptosis of HepG2 cells through an intracellular calcium increase associated with an increase in reactive oxygen species (ROS) production by the mitochondria [55].

Other channels could be used as therapeutic targets, such as the voltage-gated potassium channel. Wang et al. have demonstrated that 4-aminopyridine (4-AP) induces apoptosis of human acute myeloid leukaemia cells by raising [Ca2+]i through the P2X7 receptor pathway. 4-AP, a voltage-gated potassium channel blocker, has been shown to exert critical proapoptotic properties in various types of cancer cells, by inducing a significant increase in [Ca2+]i, through the P2X7 receptor. This calcium overload leads to disruption of the mitochondrial membrane potential and activation of caspase 3 and 9 [56].

A recent study, using xenografts of colorectal cancer treated with Sulindac and Celecoxib (nonsteroidal anti-inflammatory drugs), showed the ability of these compounds to increase [Ca2+]i, one of the consequences of which is activation of the intrinsic apoptotic pathway through the activation of calpain 9 [57].

Pumps and chemotherapies

In order to induce an increase in [Ca2+]i, two strategies are possible. The first is to target pumps expressed in the plasma membrane (PMCA) to decrease the exit of calcium from the cell, and the second is to target pumps expressed on the ER (SERCA) to deplete the calcium store.

Muscella et al. decided to decrease the activity of the PMCA pump, whose function is to extrude calcium from the cell. Authors have also synthesized a novel platinum (II) complex, [Pt(O,O′-acac)(γ-acac)(DMS)], with ~ 100-fold greater ability to induce apoptosis in endometrial cancer cells (HeLa) than cisplatin (a chemotherapeutic agent that is widely used in clinics) as well as high cytotoxicity in cisplatin-resistant MCF-7 breast cancer cells. In MCF-7 cells, drug action mechanisms include a reduction in PMCA activity, without affecting SERCA or secretory pathway Ca2+-ATPase activities. A second mechanism is the increase in calcium membrane permeability, resulting in the overall increase in [Ca2+]i that apparently facilitates a triggering of rapid calcium-dependent apoptosis [58]. Pande et al. suggested using caloxins, which are short peptides that specifically inhibit PMCA by binding to the allosteric sites on the protein's extracellular domains [59]. Using caloxins, authors have been able to increase the calcium concentration in the cell and to promote the activation of apoptotic calcium-dependent pathways. Baggott et al. have demonstrated that disruption of the PMCA2–calcineurin interaction in a variety of human breast cancer cells results in the activation of the calcineurin–NFAT pathway, and finally to the upregulation of proapoptotic protein expression [31].

To induce an increase in calcium in the cell, some teams have decided to focus on the SERCA pump. Thapsigargin, extracted from the umbelliferous plant Thapsia garganica, is a specific inhibitor of SERCA pumps. When SERCA pumps are inhibited, the ER is slowly depleted by leak channels, leading to a decrease in ER content, the consequences of which are the induction of ER stress [60] and the activation of SOCE [39]. Both of these processes could induce apoptosis. Thapsigargin is cytotoxic, which is why it has been targeted as a potential therapeutic agent. Thapsigargin has been clinically trialled in combination with a peptide target. These peptide targets include molecules on the plasma membrane of prostate cells, such as protein surface antigen [61] and prostate surface membrane antigen [62]. The development of more efficient analogues is in progress, to improve this thapsigargin in association with peptide approach, by obtaining better affinity and specificity for the SERCA pump [63].

Chemotherapy based on calcium homeostasis – triggering calcium flux and ER stress

Apoptotic calcium signalling involves two critical factors, namely the sustained elevation of [Ca2+]i, and a prolonged decrease in [Ca2+]ER, with variable contributions from the two factors, depending on cell type.

Some anticancer drugs such as cisplatin, which are widely used in clinics, are still based on triggering calcium homeostasis. Cisplatin is the first member of a class of platinum-containing anticancer drugs, which now also includes carboplatin and oxaliplatin; another is tamoxifen (an antagonist of the oestrogen receptor in breast tissue).

Cisplatin is a chemotherapeutic agent that is generally recognized as a DNA-damaging drug; its death-inducing signalling is still poorly understood. A recent study provided evidence that cisplatin induces apoptosis by activating the proapoptotic BH3-only protein bid after calpain activation by calcium, thereby contributing to cytochrome c release and subsequent caspase activation [64].

The calcium signalling induced by tamoxifen has been more extensively described. This chemotherapeutic agent is able to enhance calcium signalling in both breast cancer and several primary glioma cells, increasing both the radius of calcium wave propagation by local stimulation and the amplitude of agonist-induced calcium elevations, while retarding the normalization of cytosolic calcium [65].

The activation of the mitochondrial pathway inducing apoptosis is often targeted by chemotherapeutic agents. ROS are molecular actors that partially mediate mitochondrium-dependent apoptosis.

In human glioblastoma cells, Liang et al. have proposed the use of carvacrol to induce a rise in [Ca2+]i by inducing phospholipase C-dependent calcium release from the ER and calcium entry via protein kinase C-sensitive, non-store-operated calcium channels. These authors assume that carvacrol-induced cell death might involve ROS-mediated apoptosis [66].

A novel gallium complex, GaQ(3) (KP46), earlier developed by Valiahdi et al., is currently in phase I of clinical trials. GaQ(3) induces apoptosis via caspase/poly(ADP-ribose) polymerase cleavage in a variety of cancers. GaQ(3) also triggered intracellular calcium release in MCF-7, H1299 and PC3 cells. This calcium release promotes the stabilization of a p53–p300 complex and the recruitment of p53 to the p53 promoter, leading to p53 mRNA and protein synthesis. p53 induced more intracellular calcium release and led to ROS signalling. ROS also increased membrane translocation of FAS and FAS-mediated extrinsic apoptosis [67].

Diospyrin, a naphthoquinonoid, and its diethylether derivative (D7) increased the concentration of calcium in the cytoplasm in MCF7 cells, by inducing sustained release of calcium from ER stores via oxidative stress and phosphotidylcholine-specific phospholipase C (PC-PLC) activation. Furthermore, this elevated cytosolic calcium concentration has been found to activate apoptotic proteases, such as calpain and caspase 12, which participate in the execution of apoptosis in human breast cancer cells, involving a loss of mitochondrial membrane potential and cytochrome c release [68].

A promising pathway for inducing apoptosis of cancer cells is the activation of ER stress. On the basis of this hypothesis, cryptotanshinone, the major active constituent isolated from the root of Salvia miltiorrhiza Bunge, has been identified as a potent stimulator of ER stress. This activation of ER stress leads to apoptosis in many cancer cell lines, including HepG2 hepatoma and MCF7 breast carcinoma cells, by the activation of the mitochondrial pathway and the generation of ROS [69].

The activation of ER stress seems to be a potential alternative where resistance to chemotherapeutic agents has emerged. Vismodegib (GDC-0449), an orally administered small molecule with antineoplastic activity, has been shown to promote apoptosis of cells that are resistant to classical anticancer drugs such as cisplatin. It has been demonstrated that this new compound has the ability to induce two calcium-dependent proapoptotic factors: an increase in [Ca2+]i and a reduction in [Ca2+]ER [70].

Some cancers can be characterized by an increase in the expression of Bcl-2 protein [4], and it has been extablished that Bcl-2 can interact with IP3R [71]. On the basis of these two facts, Rong et al. decided to design a peptide corresponding to the Bcl-2-interacting region in the regulatory and coupling domain of IP3R. Their results have shown that this peptide reverses the inhibitory effect of Bcl-2, and can restore apoptosis by inducing calcium elevation. In this way, they demonstrated that the interaction of Bcl-2 with IP3Rs contributes to the regulation of proapoptotic calcium signals by Bcl-2, and that such a peptide could be used as an apoptosis inducer [72].

Conclusion

Calcium-transporting proteins, which are involved in the cellular process of apoptosis, still constitute a novel area of research in oncology. As this field is relatively new, not all aspects of oncogenic calcium homeostasis have been investigated, and its roles in different types of cancer is only just beginning to be understood. New studies of calcium-transporting proteins are necessary to allow the development of new chemotherapies, as are fresh models and technical approaches.

Acknowledgements

The research of N. Prevarskaya and F. Vanden Abeele is supported by grants from Inserm (Institut National de la Santé et de la Recherche Médicale), Ligue Nationale Contre le Cancer, FRM (Fondation de Recherche Medicale), ARC (Association pour la Recherche sur le Cancer), and Région Nord/Pas-de-Calais. C. Dubois is a recipient of a PhD stipend from the Faculty of Health Science, Aarhus University (DK) and from the Faculty of Biology and Health, Lille 1 University (FR).

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